Department of Chemistry, Zhejiang University, Hangzhou, 310027, Zhejiang, China.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, Liaoning, China.
Anal Bioanal Chem. 2019 Jul;411(18):4167-4173. doi: 10.1007/s00216-019-01819-w. Epub 2019 Apr 15.
Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.
由于寡核苷酸的磷酸二酯骨架带高度负电荷，通常采用离子对反相液相色谱（IP-RPLC）与负离子模式电喷雾电离质谱（ESI-MS）联用的方法进行分析。在此，通过提高正离子模式 ESI-MS 的源内能量，大大缓解了三乙胺（TEA）加合物的信号抑制效应，从而改善了离子对试剂 TEA/六氟异丙醇（HFIP）的效果。该策略通过分析其甲酸水解物，应用于不同的 RNA 测序，通过 IP-RPLC-MS 进行分析。与负离子模式相比，我们证明了正离子模式的 IP-RPLC-MS 分析更适合 RNA 测序，因为它的污染物干扰较少。最后，通过正离子模式 IP-RPLC-MS 分析实现了寡核苷酸和蛋白质消化物的在线同时分离和检测，彼此之间几乎没有干扰。
